Oligonucleotide-polyamide conjugates

ABSTRACT

This invention relates to novel oligonucleotide-polyamide conjugates preferably having a free 3&#39; hydroxl moiety, and wherein the polyamide is coupled to the oglionucleotide through its carboxyl terminus. A nucleotide polymer conjugate of the formula (I): Nu--NUC--C═C--X 1  --NH--X 2  --X 3  where X 1  is an unsubstituted or substituted C 1  -C 10  alkylene group, in which one or more carbons may optionally be replaced by --NH--, --O-- or --S--; X 2  is a bond, or an unsubstituted or substituted C 1  -C 20  alkylene group, in which one or more carbons may optionally be replaced by --NH--, --O-- or --S--; the optional substituents in X 1  or X 2  are selected from a variety of groups; X 3  is an amino acid or a polyamide linked via its carboxy terminus; NUC is a nucleoside group of any one of formulas (a), (b), (c), (d) where→indicates the bond to the --C--C-- group in formula (I), and X 4  is a sugar group of formula(e) where the 5&#39; oxygen is linked to Nu, and X 5  and X 6  are each independently H or OR, where R is H, a protecting group, or a solid phase matrix, and Nu is an oligonucleotide. Methods of preparing these compounds, and various uses, for example, as PCR primers, or as a substrate for DNA or RNA polymerase, are also disclosed.

This application is a national stage of PCI/AU93/00252 filed May 28,1993 and a continuation-in-part of application Ser. No. 08/068,604 filedMay 27, 1993, now U.S. Pat. No. 5,552,540 which is a continuation ofapplication Ser. No. 07/457,747 filed Dec. 22, 1989 (abandoned).

TECHNICAL FIELD

This invention relates to novel oligonucleotide-polyamide conjugatespreferably having a free 3' hydroxyl moiety, and wherein the polyamideis coupled to the oligonucleotide through its carboxyl terminus.

BACKGROUND ART

A number of molecular biological methods involve the detection ofnucleotide sequences, or the tagging of nucleotide sequences withreporter groups. For example, the widely utilised polymerase chainreaction technique (PCR) allows many copies of a desired DNA to begenerated from only a few target molecules within a period of 1 to 2hours and consequently has found applications of nucleic acid detectiontechniques previously limited by low sensitivity. Detection of amplifiedPCR products may be carried out in a number of ways. It is mostdesirable to be able to detect amplification products non-radioactively,for example, using a reporter group within the amplified nucleotidesequences.

Similarly, in reactions such as nick translation, it is desirable toincorporate a labelled nucleotide into reaction products produced by DNApolymerase or RNA polymerase for subsequent detection of hybridisationproducts.

DISCLOSURE OF INVENTION

In accordance with the present invention there is provided in one aspecta nucleotide polyamide conjugate of the formula (I):

    Nu--NUC--C═C--X.sup.1 --NH--X.sup.2 --X.sup.3          (1)

where, X¹ is an unsubstituted or substituted C₁ -C₁₀ alkylene group, inwhich one or more carbons may optionally be replaced by --NH--, --O-- or--S--, X² is a bond, or an unsubstituted or substituted C₁ -C₂₀ alkylenegroup, in which one or more carbons may optionally be replaced by--NH--, --O-- or --S--, the optional substituents in X¹ or X2 selectedfrom one or more of: oxo, amino, thioxo, hydroxyl, mercapto, carboxyl,halogen, lower alkyl, phenyl, amino-lower alkyl, ester-lower alkyl,amido-lower alkyl, ether-lower alkyl, or thioether-lower alkyl, groups,the sulfur analogues of these substituents, or the side-chainsubstituents from naturally occurring amino acids, and the closelyrelated analogues of these sidechains, for example. X³ is an amino acid,or a polyamide linked via its carboxy terminus, NUC is a nucleoside ofany one of the formulas: ##STR1## where →indicates the bond to the--C.tbd.C-- group in formula (I), and X⁴ is a sugar group of theformula: ##STR2## where the 5' oxygen is linked to Nu, and X⁵ and X⁶ areeach independently, H or OR, where R is H, a protecting group, or asolid phase matrix, and Nu is an oligonucleotide.

X¹ is an unsubstituted or substituted C₁ -C₁₀ alkylene group, in whichone or more carbons may optionally be replaced by --NH--, --O-- or--S--, and the optional substituents in X¹ are selected from one or moreof oxo, amino, thioxo, hydroxyl, mercapto, carboxyl, halogen, loweralkyl, phenyl, amino-lower alkyl, ester-lower alkyl, amido-lower alkyl,ether-lower alkyl, or thioether-lower alkyl, groups, and the like, suchas the sulfur analogues of these compounds, and any other functionalgroups. Other possible substituents are the side-chain substituents fromnaturally occurring amino acids, and their closely related analogues,for example. Preferably, the C₁ to C₁₀ alkylene is C₁₋₃ alkylene and isoptionally substituted with one or more of amide, halogen, aryl, esterand the like. A preferred form of X¹ is methylene.

X² is a bond, or an unsubstituted or substituted C₁ -C₂₀ alkylene group,in which one or more carbons may optionally be replaced by --NH--, --O--or --S--, and the optional substituents in X² are selected from one ormore of oxo, amino, thioxo, hydroxyl, mercapto, carboxyl, halogen, loweralkyl, phenyl, amino-lower alkyl, ester-lower alkyl, amido-lower alkyl,ether-lower alkyl, or thioether-lower alkyl, groups, the sulfuranalogues of these substituents, or the side-chain substituents attachedto the α-carbon of naturally occuring amino acids, and their similaranalogues, for example. X² preferably has the form --CO--(C₁ to C₉alkylene)--NH--, where the alkylene may be further substituted, forexample with substituents attached to naturally occurring amino acids,or the like. A preferred form of X² is --CO--(CH₂)₅ --NH--, or the like.

The polyamide (X³) is preferably a polypeptide comprising two or moreamino acids. Preferably, the polyamide also contains one or morereporter groups. Alternatively, the polyamide may comprise a singleamino acid.

The nucleoside group NUC in one preferred form has the structure:##STR3## where X⁴ is as described before. In the sugar moiety, X⁴,substituents X⁵ and X⁶ are each independently, H or OR, where R is H, aprotecting group, or a solid phase matrix, and preferably R⁵ is H, andR⁶ is OR, where R is H or a support matrix.

Particularly preferred compounds of this invention comprise compounds ofthe formula (II): ##STR4## wherein X², X³, X⁵, X⁶ and Nu are aspreviously described.

The group X³ represents a polyamide which is linked covalently to thegroup X². The polyamide may be formed from naturally occurring aminoacids (Biochemistry, 2nd Edition, Albert L. Lehninger, pp. 72-77, 1970),such as lysine, valine, glycine, serine, threonine, tyrosine,methionine, proline, etc. linked through amide or so-called peptidebonds. Alternatively, the polyamide may be formed from synthetic aminoacids, synthetic amino acids being those amino acids which do not occurnaturally in proteins, or else the polyamide may be a combination ofnatural and synthetic amino acids. Preferably, the synthetic amino acidsmay comprise α,ω-amino-carboxylic acids which may be represented by thegeneral formula H₂ NCHR¹ COOH, where R¹ is any organic moiety such asC₁₋₂₀ alkylene which may be straight- or branch-chained, eithersaturated, or unsaturated by having one or more olefinic or acetylinicC--C bonds for example, cycloalkyl, which may be saturated or partiallysaturated and/or interrupted by one or more hetero atoms or groupscontaining such hetero atoms such as amide groups and/or substitutedwith halogen, cyano, amino or unsubstituted or substituted phenyl orbenzyl, as just some examples. The polyamide may contain any number ofamino acid units (residues) for example from 1 to 100 amino acids.

The polyamide (X³) may form a peptide comprising naturally ornon-naturally occurring amino acids. The sequence of the peptide can bedesigned to suit any desired application, such as interaction withantibodies, enzymic reactions and the like.

The polyamide may contain one or more reporter groups attached to thepolyamide chain via a derivatised amino acid such as lysine. Reportergroups may comprise fluorescent moieties, chemiluminescent moieties,paramagnetic moieties and the like, biotin and colloidal compounds suchas ferritin or colloidal silver or gold and enzymes. Reporter groups maybe covalently linked one or more amino acids within the polyamide,particularly through the free amino group of lysine.

Fluorophore reporter groups may be selected from:fluorescein-5-isothiocyanate, diacyl (such as isobutyryl, acetyl orpivaloyl) fluorescein-5 and/or 6 carboxylic acid pentafluorophenylester, 6-(diacyl-5 and/or 6-carboxamide-fluorescein)amino-hexanoic acidpentafluorophenyl ester, Texas Red (Trademark of Molecular Probes,Inc.), tetramethylrhodamine-5 (and 6) isothiocyanate,oesin-isothiocyanate, erythrosin-5-isothiocyanate,4-chloro-7-nitrobenz-2-oxa-1,3-diazole,4-fluoro-7-nitrobenz-2-oxa-1,3-diazone,3-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) methylamino-propionitrile,6-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)aminohexanoic acid, succinimidyl12-(N-methyl-N-(7-nitrobenz-2-oxa-1,3-diazol-4-71)aminododecanoate,7-diethylamino-3-(4'-isothio-cyanatophenyl)-4-methylcoumarin (CP),7-hydroxycoumarin-4-acetic acid, 7-dimethylamino-coumarin-4-acetic acid,succinimidyl 7-dimethylaminocoumarin-4-acetate,7-methoxycoumarin-4-acetic acid,4-acetamide-4'-isothiocyanatostilbene-2-2'-disulfonic acid (SITS),9-chloroacridine, succinimidyl 3-(9-carbazole)-propionate, succinimidyl1-pyrenebutyrate, succinimidyl 1-pyrenenonanoate, p-nitrophenyl1-pyrenebutyrate, 9-anthracenepropionic acid, succinimidylanthracene-9-propionate, 2-anthracenesulfonyl chloride; or fluorophoreprecursors, which when treated in a particular manner fluoresce.

Reporter groups may be attached to polyamides according to conventionaltechniques known per se in the art. For example, nucleophilic groups onpolyamides such as primary amine groups may react with the fluorescentor enzymic reporter groups to form a covalent bond therebetween.Alternatively, bifunctional coupling reagents known per se in the art(for example as described in the Pierce Chemical Company catalogue,1987) may be employed to attach reporter groups to polyamides

Biotin may be incorporated into the polyamide by conventional methods.For example, underivatised biotin may be incorporated into a polyamideutilising the BOP coupling method (Castro, B, et al., Synthesis (1976)pp. 751-752). Alternatively, biotin can be introduced as theN-hydroxysuccinimidyl active ester. It may also be incorporated by usinga biotinylated amino acid derivative, for instance. Biotin may bedetected using avidin attached to a reporter group. For example,streptavidin-alkaline phosphatase conjugate may be employed to bind tobiotin. The alkaline phosphatase can react with a suitable substrate togenerate an insoluble diprecipitate which can be detected visually.

Enzymic reporter groups may be selected from β-galactosidase, horseradish peroxidase, urease, alkaline phosphatase, dehydrogenases,luciferase and carbonic anhydrase. Generally, enzymes will react withone or more substrates to produce a detectable signal such as a colourchange, luminescence or formation of a precipitate.

The number of reporter groups which may be included in the polyamide isunimportant to this invention, and for example, from 1 to 20 or morereporter groups may be incorporated into the polyamide. The positioningof the reporter groups within the polyamide is not important to thisinvention. For example, a single reporter group may be present at theterminal end of the polyamide distal to the alkyne amino group.Alternatively, a reporter group may be proximal to the alkyne aminogroup. As a further alternative, multiple reporter groups may bedistributed along the length of the polyamide.

The oligonuceotide Nu is any suitable nucleotide sequence, but in onepreferred form has the general formula: ##STR5## where B isindependently selected from adenyl, guanyl, thyminyl or cytosinyl, and nis from 1 to about 400, or more preferably from 2 to about 200.

The oligonucleotide sequence Nu which extends from the 5' hydroxylmoiety of the sugar residue compounds of the formulae I and II may be ofany desired nucleotide sequence and composition which allowshybridisation to a DNA or RNA target and further is capable of acting asa primer for DNA or RNA polymerase. The oligonucleotide may be comprisedof deoxyribonucleotides, ribonucleotides or a combination of deoxy andribonucleotides. The oligonucleotide may comprise from 1 to 400nucleotides or more, preferably 2 to 200 nucleotides. Theoligonucleotides may be suitably modified to increase half-life in-vivowithout effecting hybridisation. For example, the oligonucleotide may bemodified by replacing 1 or more of the non-bridging oxygens on thephosphorous backbone with sulphur or amines, according to the proceduresof Argawal et al. (Proc. Nail. A cad. Sci. USA 85 (1988), pp. 7079-7083)or Stein and Cohen, (Cancer. Res. 48 (1988), pp. 2659-2688). Suchmodified oligonucleotides are within the scope of the termoligonucleotide. The term "oligonucleotide" may also include a singlenucleotide (ribo or deoxyribonucleotide) or a polynucleotide comprisesof ribonucleotides, deoxyribonucleotides or mixtures thereof.

The general process for preparing a nucleotide polymer conjugate of theformula (I)

    Nu--NUC--C═C--X.sup.1 --NH--X.sup.2 --X.sup.3          (I)

where, the substituents are as described above, involves a processcomprising: (1) providing a compound of the formula (III): ##STR6## inwhich, NUC' is a group having any one of the formulas: ##STR7## and X¹,X⁵, and X⁶ are as previously described, and Pr¹ and Pr² are protectinggroups which may be the same or different; (2) deprotecting the pendantamino group by removing Pr¹ in compound (III) under conditions which mayor may not remove Pr² and thereafter reacting the deprotected compoundwith a compound of the formula Pr³ X² R^(X) wherein X² is as previouslydescribed, Pr³ is a protecting group and R^(x) is a leaving group, so asto covalently link X² to the pendant amino group, to give: ##STR8## andin the case where the 5'-OH group is free this group is optionallyreprotected with Pr² a removable protecting group, the same or differentto Pr² in step (1), or when X² is a bond omitting step (2);

(3) deprotecting the pendant amino group by removing Pr³ (or Pr¹ when X¹is a bond) in the compound of step (2) and reacting it with an activatedamino acid or polyamide, to introduce all or part of X³, and if onlypart of X³ has been introduced, thereafter sequentially adding one ormore activated amino acids or polyamides one or more times understandard peptide synthesis conditions to add the remainder of X³ to thecompound, to form: ##STR9##

(4) deprotecting the 5'-OH group of the sugar moiety of the compound ofstep (3) if not previously deprotected and reacting the deprotected OHgroup with an activated nucleotide or oligonucleotide to form a 5'-3'bond, and thereafter sequentially adding one or more activatednucleotides to form an oligonucleotide chain, to add Nu to the compound;and (5) optionally removing any remaining protecting groups, andoptionally cleaving said compound from a solid phase matrix where X⁵ orX⁶ is OR and R is a solid phase matrix, to give compound (I).

Amino and hydroxy groups on compounds of the formulae I and II may beprotected with suitable protecting groups such as described by Green(Protecting Groups in Organic Synthesis, John Wiley & Sons, Inc., 1981).For example, hydroxy protecting groups may be selected from acyl such assubstituted or unsubstituted alkanoyl (e.g. formyl, acetyl, propionyl,butyryl, isobutyryl, valeryl, bromoacetyl, dichloroacetyl,trifluoroacetyl), substituted or unsubstituted aroyl (e.g. benzoyl,toluoyl, xyloyl, nitrobenzoyl, bromobenzoyl, salicyloyl), arylalkyl(e.g. benyl), methyl, methoxy, methylthiomethyl,2-methoxyethoxymethyl,bis(2-chloroethoxy)methyl,tetrahydropyranyl,tetrahydrothiopyranyl,4-methoxytetrahydropyranyl, 4-methoxytetrahydrothiopyranyl,tetrahydrofuranyl, tetrahydrothiofuranyl, 1-ethoxyethyl,1-methyl-1-methoxyethyl 2-(phenylselenyl)ethyl, t-butyl, allyl, benzyl,o-nitrobenzyl, triphenylmethyl, α-naphthyldiphenylmethyl,p-methoxyphenyldiphenylmethyl, 9-(9-phenyl-10-oxo)anthryl (Tritylone),dimethoxy trityl or pixyl, trimethylsilyl, isopropyldimethylsilyl,t-butyldimethylsilyl, triisopropylsilyl. Amino protecting groups may beselected from acyl, particularly organic acyl, for example, substitutedor unsubstituted aliphatic hydrocarbonoxycarbonyl such as alkoxycarbonyl(e.g. methoxycarbonyl, ethoxycarbonyl, propoxycarbonyl, butoxycarbonyl,t-butyloxy-carbonyl. 5-pentoxycarbonyl); haloalkoxycarbonyl (e.g.chloromethoxy-carbonyl tribromoethoxycarbonyl, trichloroethoxycarbonyl);an alkane- or arene- sulfonylalkoxy-carbonyl (e.g.2-(mesyl)ethoxycarbonyl, 2-(p-toluenesulonyl)-ethoxycarbonyl); analkylthio-orarylthioalkoxycarbonyl(e.g.2-(ethylthio)ethoxycarbonyl,2-(p-tolylthio)-ethoxycarbonyl),substituted or unsubstituted alkanoyl such as halo(lower)alkanoyl (e.g.formyl, trifluoroacetyl); a monocyclic or fused cyclic-alicyclicoxycarbonyl (e.g. cyclohexyloxycarbonyl, adamantyloxycarbonyl,isobomyloxycarbonyl); substituted or unsubstituted alkenyloxycarbonyl(e.g. allyoxycarbonyl); substituted or unsubstituted alkynyloxycarbonyl(e.g. 1,1-dimethylproparglyoxycarbonyl); substituted or unsubstitutedaryloxycarbonyl (e.g. phenoxycarbonyl, p-methylphenoxycarbonyl);substituted or unsubstituted aralkoxycarbonyl (e.g. benyloxycarbonyl,p-nitrobenzyloxycarbonyl, p-phenylazobenzyloxycarbonyl,p-(p-methoxyphenylazo)-benzyloxycarbonyl, p-chlorobenzyloxycarbonyl,p-bromobenzyloxycarbonyl, α-naphthylmehoxycarbonyl,p-biphenylisopropoxycarbonyl, fluorenymethoxycarbonyl); substituted orunsubstituted arenesulfonyl (e.g. benzenesulfonyl,p-toluenesulfonyl);substituted or unsubstituted dialkylphosphoryl (e.g.dimethylphosphoryl); substituted or unsubstituted diaralkylphosphoryl(e.g. O,O-dibenzylphosphoryl); substituted or unsubstitutedaryloxyalkanoyl (e.g. phenoxyacetyl, p-chlorophenoxyacetyl,2-nitrophenoxyacetyl, 2-methyl-2-(2-nitrophenoxy)propyonyl); substitutedor unsubstituted aryl such as phenyl, tolyl; or substituted orunsubstituted aralkyl such as benzyl, diphenylmethyl, trityl ornitrobenzyl.

Particularly preferred protecting groups are 4,4'-dimethoxytrityl, Fmoc,BOC and Pixyl.

The above protecting groups may comprise Pr¹ and Pr² (or Pr³) asreferred to hereinafter The above protecting groups may also be used toprotect X⁵ or X⁶.

Compounds of the formulae I and II may be utilised to extend a templatenucleotide sequence utilising DNA polymerase, such as the Klenowfragment or RNA polymerase (such as SP6 or T7 polymerase). Inparticularly, but in no way limiting this invention, the compounds ofthe formulae I and II may be utilised in the polymerase chain reaction(PCR) where the nucleotide sequence of the group Nu is selected to becomplementary to a portion of a target sequence. Annealing of selectedoligonucleotide "primers" to complementary sequences on opposite strandsof a target DNA at low temperature followed by extension in a 3'direction by a thermo stable polymerase results in gene amplification.The amplified products can be readily detected by virtue of reportergroups containing within the polyamide residue. For example, fluorescentreporter groups may be detected by irradiating the amplified productswith a light source within the excitation frequency of the fluorophore.Biotin containing reporter groups may be detected by reaction withavidin.

Compounds of this invention may be attached to a support matrix via thegroups X⁵ or X⁶. The support matrix may, for example, be selected fromcontrolled pore glass, such as aminopropyl controlled pore glass(AP-CPG) or polystyrene resins.

The compounds of this invention may be fully protected utilisingprotecting groups as described herein and attached to a support matrix,in a protected form but detached from a support matrix, or in a fullydeprotected form.

Compounds of the formula II ##STR10## where X², X³, X⁵, X⁶ and Nu are asdefined previously, may be prepared generally by:

(1) providing a compound of the formula (IIIa): ##STR11## in which X⁵and X⁶ are as previously described, and Pr¹ and Pr² are protectinggroups which may be the same or different;

(2) deprotecting the pendant amino group by removing Pr¹ in compound(IIa) under conditions which may or may not remove Pr² and thereafterreacting the deprotected compound with an amino acid of the formula Pr³X² R^(x) wherein X² is as previously described, Pr³ is a protectinggroup and R^(x) is a leaving group, so as to covalently link X² to thependant amino group, and in the case where the 5'-OH group is free thisgroup is optionally reprotected with a removable protecting group whichmay be the same or different to the protecting group Pr² in step (1), orwhen X² is a bond omitting step (2); (3) deprotecting the pendent aminogroup by removing Pr³ (or Pr¹ when X² is a bond) in the compound of step(2) and reacting it with an activated amino acid or polyamide, tointroduce all or part of X³, and if only part of X³ has been introduced,thereafter sequentially adding one or more activated amino acids orpolyamides one or more times under standard peptide synthesis conditionsto add the remainder of X³ to the compound; (4) deprotecting the 5'-OHgroup of the sugar moiety of the compound of step (3) if not previouslydeprotected, and reacting the deprotected OH group with an activatednucleotide or oligonucleotide to form a 5'-3' bond, and thereaftersequentially adding one or more activated nucleotides to form anoligonucleotide chain, to add Nu to the compound; and (5) optionallyremoving any remaining protecting groups, and optionally cleaving saidcompound from solid phase matrix where X⁵ or X⁶ is OR and R is a solidphase matrix, to give compound (II).

In the synthesis of compounds of the formula I and II where X² is abond, step (2) comprises deprotecting the pendant amino group, withoptional deprotection of the 5'OH group, and reacting the deprotectedamino group with X² and then with X³, as described above, each of whichmay be, for instance, an activated amino acid or peptide so as tocovalently link the amino acid to the pendant amino group, withoutaddition to the protected or deprotected 5'OH group of compounds of theformula (III); and in the case where the 5'OH group of compounds of theformula (III) is free this group is optionally reprotected with aremovable protecting group.

Polyamides X³ may, for example, be synthesised using solid phase Fmoc(Atherton, R. and Sheppard, R. C. (1985) J Chem. Soc. Commun., pp.165-166) or solid phase Boc (Barany, G. and Merrifield, R. B. (1980)Solid-Phase Peptide Synthesis in "The Peptides", Vol. 2, E. Gross & J.Meienhofer Eds., Academic Press, New York, pp. 1-284) methodologies. Inthese methods, the amino acids are protected with standard protectinggroups known per se in the art (for example, Green (1981) ProtectiveGroups in Organic Synthesis, John Wiley & Sons, Inc.; Atherton andSheppard (1985) J. Chem. Soc. Commun., pp. 165-166; Barany andMerrifield, Supra) to protect reactive moieties.

Oligonucleotides Nu may be synthesized by the solid phasephosphotriester method (Sproat and Gait (1984) OligonucleotideSynthesis, A Practical Approach, pp. 83-116, IRL Press, Oxford), solidphase H-phosphonate method (Froehler et al. (1986) Nucleic AcidsResearch 14, pp. 5399-5407) or the solid phase phosphoramidite method(Beaucage and Caruthers (1981) Tetrahedron Let., 22, pp. 1859-1862). Ineach of these methodologies, reactive groups such as hydroxy or aminogroups may be protected with standard hydroxy and amino protectinggroups as described by (Green (1981) Protective Groups in OrganicSynthesis, John Wiley & Sons, Inc.; Beaucage, S. L. and Caruthers, M. H.(1981) Tetrahedron Lett., 22, pp. 1859-1862; Sproat, S. and Gait, M. J.(1984) Oligonucleotide Synthesis, A Practical Approach, pp. 83-116, IRLPress, Oxford).

Preferably, one of the groups X⁵ or X⁶ of compounds of the formula IIIis attached to a solid phase matrix. Most preferably the group X⁶ iscoupled to a solid phase matrix. The matrix may be activated to includesuitable reactive groups as previously described. One or more reportergroups may be introduced into the polyamide at a number of differentstages. The reporter group can be present in the amino acids prior topolyamide synthesis (step 3); introduced after polyamide synthesis (step4); after oligonucleotide synthesis (step 5); or after deprotection andpurification of the oligonucleotide-polyamide conjugate. The methodchosen will depend upon the choice of reporter groups and syntheticprocedure.

If the reporter group is stable to the conditions of both peptide andoligonucleotide synthesis, it can be incorporated from the start ofpolyamide synthesis, as a derivatized amino acid. If it is stable to theconditions of DNA synthesis but not those of peptide synthesis, it canbe incorporated after the polyamide has been synthesized. If thereporter group is not stable to either peptide or oligonucleotide chainassembly, but is stable to deprotection methods, it can be incorporatedafter oligonucleotide chain assembly of the fully protectedpolyamide-oligonucleotide conjugate. If the label is not stable to anyof the conditions used in the synthesis of the compounds of theinvention, it may be introduced in a solution phase reaction with thepurified fully deprotected polyamide-oligonucleotide conjugate.

Fluorophores may be introduced into the oligonucleotide-polyamideconjugate at any of steps (3) to (6). This is also the case for biotin.

Enzymes, and colloidal compounds such as colloidal gold, colloidalsilver, ferritin, or biotin may be introduced at steps (3) to (6).

The polyamide portion of the oligonucteotide-polyamide conjugate maycontain multiple reporter groups which may increase the detectablesignal produced therefore facilitating detection.

The polyamide portion of the conjugate not only functions as a vehiclefor attaching a reporter group, but may also act as an address marker totarget a polyamide to a particular cell type, cellular location, orenhance the passage of an oligonucleotide through a cellular membrane.The address label activity of peptide sequences is well established(Verner and Schatz (1988) Science 241, pp. 1307-1313; and Goldfarb etal. (1986) Nature 322, pp.641-644). By selecting a peptide sequencewhich is, for example, recognised by a cell surface receptor,oligonucleotides conjugated to that peptide sequence may be transportedinto specific cell types where they can exert a biological effect, suchas, in the case of anti-sense oligonucleotides, blocking transcriptionof viral or cellular RNA.

In a particularly preferred method of this invention, a compound of theformula (IIIa): ##STR12## wherein Pr¹, Pr², X⁵ and X⁶ are as previouslydescribed; is attached to a solid support by the 3'-hydroxyl group (ie,X⁶ is OR, and R is a solid phase matrix); a spacer of the formula Pr³HNX² COR^(x) or amino acid is added to the deprotected pendant aminogroup as described above, followed by addition of a polyamide chain bythe sequential addition of one or more amino acid groups according toconventional peptide synthetic techniques.

An oligonucleotide chain is then added to the deprotected 5'-hydroxygroup of the compounds of the formula IV. The various protecting groupsPr¹, Pr² and Pr³ may be the same or different and are as previouslydescribed.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 herewith shows PAGE of 5'-end labelled oligonucleotides. Lanes Aand C have normal oligonucleotides and lane B the 23 mer conjugatecontaining an Ahx-Lys(Biotin)-Ala peptide.

FIG. 2 shows an outline (SCHEME I) for producing compounds according tothe present invention.

FIG. 3 shows detailed description (SCHEME II) for making the reactantsin FIG. 2.

FIG. 4 shows the first part of detailed description (SCHEME III) forproducing preferred compounds of the present invention.

FIG. 5 shows the second part of the more detailed description in SCHEMEIII, which follows on from FIG. 4.

FIG. 6 shows more description for producing the labelled amino acidsused in the peptide synthesis of the invention.

MODES FOR CARRYING OUT THE INVENTION

Specific embodiments of this invention will now be described, by way ofthe following non-limiting examples. The compounds prepared arefrequently referred to by way of reference numbers which are shown inFIGS. 2 to 6 in the drawings.

EXAMPLE 1

MATERIALS: 3-Aminopropyne, 5-iodo-2'-deoxyuridine, biotin and3-aminopropyl-triethoxysilane were purchased from Sigma.N-(Fluoren-9-ylmethoxy-carbonyloxy)-succinimide, N.sup.α -Fmoc-L-lysine,pentafluorophenol, DCC, and Fmoc-Ala-OPfp were obtained from Auspep,Melbourne. 3-Nitropyridine-2-sulfenyl chloride was obtained fromKokusan, Tokyo. 9-Chloro-9-phenylxanthene andtetrakis(triphenylphosphine)palladium(0) were supplied by Aldrich.N-hydroxysuccinimide was obtained from Pierce. Triethylamine (purissgrade), Controlled Pore Glass (200-400 mesh, 500 Å pore size, Cat. no.27720) and diisopropylcarbodiimide (DIC) were purchases from Fluka.Reagents for the ninhydrin assay were supplied by Applied Biosystems.DMF was distilled under reduced pressure and used within 14 days.Pyridine was distilled over CaH₂ at atmospheric pressure, and storedover 5 Å molecular sieves. All other reagents were used without furtherpurification. Thin layer chromatograph was performed on Merck SG-60precoated plastic plates, and flash chromatography was performed onMerck silica gel (SG-60, 230-240 mesh). Biotin-N-hydroxysuccinimidylester was prepared as described previously¹ except that 95% ethanol/1%acetic acid/4% H2O was substituted as recrystallisation solvent.N-Fmoc-εAhx-OPfp² and N-Boc-3-aminopropyne³ were prepared according toliterature methods.

Melting points were taken on an Electrothermal apparatus and areuncorrected. NMR spectra were recorded on a JEOL GX400 spectometeroperating at 399.9 MHz for ¹ H observation and at 99.98 MHz for ¹³ Cobservation. DEPT experiments were performed with a 135° ¹ H selectionpulse. The DQF-COSY experiment was run in the phase-sensitive mode ofdata accumulation with a 356×2K data matrix and 16 transients perincrement. The spectrum was obtained after zero-filling to a 1K×1K finaldata matrix and application of an exponential weighting function in bothdirections. The HMBC experiment was run in the absolute absorption modewith a 128×2K raw data matrix and 128 transients per increment. Thespectrum was obtained after zero-filling 4 times in the t, dimension andapplication of a sine-bell weighting function in both directions. Theexperiment was run assuming a ³² C-H value of 8Hz. The numbering systemused for nucleosides 3 and 4 is outlined in Scheme I while the numberingsystem for compounds 22 and 23 is as shown in Scheme IV. IR spectra wererecorded on a Perkin Elmer 1600 series FIR, using KBr discs. UV spectrawere recorded with samples dissolved in 0.1 mM EDTA solutions, on aVarian Cary 1 spectrophotometer. Amino-acid analyses were performed on aBeckman System 6300 Amino Acid Analyser. HPLC analyses were performed ona Shimadzu LC4A instrument using a Phenomenex reverse-phase C₁₈ column(5 ODS 30, 5 μm, 60 Å pore size), with buffer A being 0.1Mtriethylammonium acetate, pH 7.0 and buffer B 0.1M triethylammoniumacetate, 30% CH₃ CN, pH 7.0.

Elemental analyses were obtained from CMAS Pty. Ltd., Melbourne. Highand low resolution FAB mass spectra were recorded on JEOL DX-300 andJEOL AX-505H instruments respectively, equipped with FAB sources.Samples for high resolution measurements were suspended in apolyethylene glycol (600)/thiolglycerollglycerol/DMSO matrix;low-resolution samples in a thioglycerol matrix. The ionisation gas inboth cases was Xe.

SYNTHESIS OF REAGENTS EXAMPLE 1(a)

3-(fluoren-9-yl-methoxycarbonyl)-amidopropyne (9)

3-Aminopropyne (359 μL, 5.25 mmol) was added dropwise to a solution ofN-(fluoren-9-ylmethoxycarbonyloxy)succinimide (FmocNHS) (1.69 g, 5.00mmol) in THF (8 mL) at 0° C. and stirred for 2 h. The solution wasallowed to warm up to room temperature and the solvent was removed undervacuum. The crude residue was dissolved in ethyl acetate (100 mL) andthe solution washed with H₂ O (3×30 mL), then dried (Na₂ SO₄).Recrystallisation from ethyl acetate gave 9 as colourless needles (1.11g, 80%), mp 129-130° C. ¹ H NMR (d₆ -DMSO): δ3.13 (t, 1H, H1, J=2.3 Hz),3.80 (dd, 2H, H3, J=5.9, 2.5 Hz), 4.23 (t, 1H, Fmoc CH, J=6.8 Hz), 4.33(d, 2H, Fmoc CH₂, J=7.1 Hz), 7.33 (ddd, 2H, Fmoc H2 and H7, J=7.6, 7.5,1.2 Hz), 7.41 (ddd, 2H, Fmoc H3 and H6, J=7.6, 7.5, 0.9 Hz), 7.71 (d,2H, Fmoc H2 and H8, J=7.6 Hz), 7.81, (t, 1H, NH, J=5.9 Hz), 7.90 (d, 2H,Fmoc H4 and H5, J=7.6Hz). ¹³ C NMR (d₆ -DMSO): δ29.8 (C3), 46.6 (FmocCH, 65.7 (Fmoc CH₂), 73.1 (Cl), 81.4 (C2), 120.1 (Fmoc C3 and C6), 125.2(Fmoc C2 and C7), 127.1 (Fmoc C4 and C5), 127.6 (Fmoc C1 and C8), 140.7(C4a and C4b), 143.8 (Fmoc C8a and C9a), 155.9 (Fmoc CO). FABMS m/z 300(M+Na), 278 (M+H). Anal. calcd for C₁₈ H₁₅ NO₂ : C, 78.0; H. 5.45; N,5.05. Found: C, 78.1; H, 5.45; N, 5.01.

EXAMPLE 1(b)

3-(3-Nitro-2-sulfenylpyridine)-aminopropyne (10)

To a stirred solution of ³ -aminopropyne (855 μL, 12.5 mmol) in DMF (20mL) was added 3-nitropyridine-2-sulfenyl chloride (0.94 g, 5.0 mmol) intwo equal portions over 0.5 h. After 1.5 h, the reaction mixture waspoured into ethyl acetate (500 mL) and washed with H₂ O (3×200 mL),dried (Na₂ SO₄), and the solvent removed under vacuum. Recrystallisationfrom MeOH/H₂ O gave 10 as red crystals (0.653 g, 62%), mp 107-9° C. ¹ HNMR (d₆ -DMSO): δ3.18 (t, 1H, H1, J=2.6 Hz), 3.77 (dd, 2H, H3, J=4.4,2.6 Hz), 5.34 (t, 1H, NH, J=4.4 Hz), 7.47 (dd, 1H, NPYS H5, J=8.3, 4.6Hz), 8.61 (dd, 1H, NPYS H6, J=8.4, 1.5 Hz), 8.90 (dd, 1H, NPYS H4,J=4.4, 1.5 Hz). ¹³ C NMR (d₆ -DMSO): δ30.7 (C3), 74.8 (C1), 81.7 (C2),120.4 (NPYS C5), 134.3 (NPYS C4), 139.8 (NPYS C3), 154.1 (NPYS C6),163.1 (NPYS C2). FABMS m/z 210 (M+H). Anal. calcd for C₈ H₇ N₃ O₂ S: C,45.9; H, 3.37; N, 20.1. Found: C, 45.7; H, 3.33; N, 20.1.

EXAMPLE 1(c)

³ -(9-Phenylxanthen-9-yl)aminopropyne (12)

9-Chloro-9-phenylxanthen (14.7 g, 50 mmol) was added in two equalportions over 0.5 h to a stirred solution of 3-aminopropyne (8.6 mL, 125mmol) in DMF (100 mL). After 3 h, MeOH (20 mL) was added and thereaction mixture was stirred for 15 min. The mixture was then extractedinto diethyl ether (500 mL) and washed with H₂ O (3×300 mL), dried (Na₂SO₄), and the solvent removed under vacuum. The resulting yellow oil wasdried under high vacuum (4 h), and then redissolved in hot diethyl ether(70 mL). The solution was kept at -20° C. for 24 h and then filtered,the solid washed with ice-cold hexand (2×50 mL) followed by ice-colddiethyl ether (2×50 mL), giving 12 as a colourless powder (12.7 g, 82%),mp 116-8° C. ¹ H NMR (d₆ -DMSO): δ2.86 (dd, 2H, H3, J=7.3, 4.4 Hz), 2.99(t, 1H, H1, J=2.4 Hz), 4.01 (t, 1H, NH, J=7.2 Hz), 6.98-7.50 (m, 13H, PxCH). ¹³ C NMR (d₆ -DMSO): δ32.8 (C3), 59.7 (Px C9), 73.4 (C1), 82.7(C2), 1160.0 and 123.5 (Px CH), 125.0 (Px C8a and C9a), 126.3, 126.4,128.0, 128.6, 128.7 (Px CH), 149.5 (Px C1'), 150.7 (Px C4a and C10a).FABMS m/z 311 (M+). Anal. calcd for C₂₂ H₁₇ NO: C, 84.8; H, 5.51;N,4.50. Found: C, 84.4; H. 5.73; N, 4.30.

EXAMPLE 1 (d)

5-Iodo-5'-O-(9-phenylxanthen-9-yl)-2'-deoxyuridine (7)

5-Iodo-2'-deoxyuridine (IDU) (3.54 g, 10 mmol) was coevaporated with drypyridine (3×20 mL). The IDU was redissolved in dry pyridine (15mL) and9-phenyl-9-chloroxanthen (3.82 g, 13 mmol) was added to the stirringsolution in two equal portions over 0.5 h. After 1h, MeOH (5 mL) wasadded and the solution stirred for a further 0.5 h. The solvent was thenremoved under vacuum, and the residue recrystallized from a 1% Et₂N/ethyl acetate solution giving 7 as colourless crystals (4.27 g, 70%),mp 200-2° C. ¹ H NMR (d₆ -DMSO): δ2.20 (m, 2H, H2'), 2.99 (dd, 1H, H5',J-10.5, 4.2 Hz), 3.10 (dd, 1H, H5", J=10.5, 2.7 Hz), 3.85 (m, 1H, H4'),4.15 (m, 1H, H3'), 5.29 (d, 1H, 3'OH, J=4.2 Hz), 6.08 (t, 1H, H1', J=7.0Hz), 7.10-7.42 (m, 13H, Px CH), 8.08 (s, 1H, H6). ¹³ C NMR (d₆ -DMSO):δ40.4 (C2'), 63.8 (C5'), 69.9 (C5), 71.0 (C3'), 75.6 (Px C9), 85.2(Cl'), 85.9 (C4'), 116.3, 116.4 (Px CH), 122.3, 122.4 (Px C8a and C9a),123.8, 124.0, 125.8, 126.8, 128.2, 129.3, 129.69, 129.7, 129.73 (Px CH),143.9 (C6), 148.4(Px Cl'), 150.0 (C2), 150.51, 150.58 (Px C4a and C10a),160.6 (C4). FABMS m/z 633 (M+Na'). Anal. calcd for C₂₈ H₂₃ N₂ P₆ I: C,55.1; H,3.81, N, 4.59. Found: C, 55.0; H, 3.81, N, 4.54.

EXAMPLE 1(e)

5-[3(-tert-Butyloxycarbonylamido)prop-1-yn-1-yl)]-5'-O-(9-phenyl-xanthen-9-yl)-2'-deoxyuridine(3)

To a degassed (Ar) solution of 11 (1.22 g, 2.00 mmol) in DMF (6mL) wasadded CuI (0.076 g, 0.40 mmol), Et₃ N (558 μL, 4.00 mmol),3-tery-butyloxycarbonylamidopropyne¹¹ (0.932 g, 6.00 mmol) and (Ph₃ P)₄Pd^(o) (0.232 g, 0.20 mmol) successively and the solution was stirredfor 5 h. AB1X8(HCO₃ --) ion-exchange resin (6 molar equiv) was addedwith MeOH (10 mL) and CH₂ Cl₂ (10 mL), and the mixture stirred for 30min. The resin was removed by filtration, and the solvent removed underreduced pressure. The crude residue was dissolved in ethyl acetate (200mL), the solution was washed with H₂ O (3×100 mL), dried (Na₂ SO₄), andfiltered. After removal of the solvent, flash silica gel chromatography(70 g silica, 0-10% MeOH/CH₂ Cl₂) followed by recrystallisation fromchloroform/diethyl ether gave 3 (0.602 g, 47%) as colourless crystals,mp 157-160° C. ¹ H NMR (d₆ DMSO): δ1.35 (s, 9H, Boc CH₃, 2.25 (m, 2H,H2'), 3.00 (dd, 1H, H5', J=10.5, 4.4 Hz), 3.12 (dd, 1H, H5", J=10.4, 2.6Hz), 3.69 (m, 2H, H9), 3.91 (m, 1H, H4'), 4.15 (m, 1H, H3'), 5.30 (d,1H, 3'OH, J=4.2 Hz), 6.09 (5, 1H, H1', J=6.7 Hz), 7.10-7.45 (m, 13H, PxCH), 7.96 (s, 1H, H6). ¹³ C NMR (d6-DMSO): δ28.2 (Boc CH₃), 30.0 (C9),40.3 (C2'), 63.8 (C5'), 70.8 (C3'), 73.8 (C8), 75.5 (Px C9), 78.2 (BocC), 85.3 (Cl'), 85.9 (C4'), 90.1 (C7, 98.4 (C5), 116.1, 116.3 (Px CH),122.0 (Px C8a and C9a), 123.9, 124.0, 125.8, 126.7, 128.1, 129.0, 129.1,129.7 (Px CH), 142.9 (C6), 148.3 (Px C2), 149.3 (C1'), 150.5, 150.7 (PxC4a and C10a), 155.1 (Boc CO), 161.6 (C4). FABMS m/z 660 (M+Na). Anal.calcd for C₃₆ H₃₅ H₃ O₈ C, 67.8; H, 5.54; N, 6.59. Found C, 67.7; H,5.68; N, 6.54.

EXAMPLE 1(f)

The Fmoc-nucleoside 1 was prepared in an analogous manner but it couldnot be purified (to analytical purity due to persistent contaminatingstarting nucleoside 7. ¹³ C NMR (CDCl₃): δ31.2 (C9), 41.8 (C2'), 46.9(Fmoc C9), 60.3 (C5'), 63.4 (C8), 66.4 (Fmoc CH2), 72.4 (C3'), 74.3 (PxC9), 86.1 (C1'), 86.7 (C4'), 89.5 (C7), 99.4 (C5), 116.4 (Px CH), 119.8(Fmoc C3 and C6), 122.1, 122.3 (Px C8a and C9a), 123.7, 123.8 (Px CH),124.8 (Fmoc C2 and C7), 126.2 IPx CH), 126.85, 126.95 (Fmoc C4 and C5),127.8 (Fmoc C1 and C8), 129.4, 129.6 (Px CH), 141.1 (Fmoc C4a and C4b),143.2, 143.6 (Fmoc C8a and C9a), 143.7 (C6), 148.1 (C2), 149.4 (Px Cl'),151.06, 151.12 (Px C4a and C10a), 155.6 (Fmoc CO), 162.25 (C4).

EXAMPLE 1(g)

5-[3-(Phenylxanthen-9-ylamino)prop-1-yn-1-yl]-5'-O-(9-phenyl-xanthen-9-yl)-2'-deoxyuridine(4)

The procedure was identical to that of 3 except that the crude residuewas redissolved in CH₂ Cl₂ (200 mL). The solution was washed with 10%NaHCO₃ (2×100 mL) and H₂ O (1×100 mL). After the solution had been dried(Na₂ SO₄), filtered, and the solvent evaporated, flash silica gelchromatography (0-5% MeOH/CH₂ Cl₂. 1% Et₃ N) gave 4 as a fawn solid(0.608 g, 76%). The portion was recrystallised from MeOH to analyticalpurity, mp 156-8° C. (decomp.). ³ H NMR (d₆ -DMSO): δ2.25 (m, 2H, H2'),2.55 (dd, 1h, H9a, J=16.3, 7.15 Hz), 2.66 (dd, 1H, H9b, J=16.3, 7.14Hz), 2.95 (dd, 1H, H5', J=10.6, 3.67 Hz), 3.07 (dd, 1H, H5", J=10.4,2.38 Hz), 3.51 (t, 1H, N9H, J=7.20Hz), 3.9 (m, 1H, H4'), 4.21 (m, 1H,H3'), 5.31 (d, 1H, 3'OH, J=4.03 Hz), 6.11 (t, 1H, H1', J=6.78 Hz),6.85-7.40 (m, 26H, NPx and OPx CH), 8.05 (s, 1H, H6), 11.6 (br s, 1H,H3). ¹³ C NMR (d₆ -DMSO): δ33.4 (C9), 40.8 (C2'), 59.6 (NPx C9), 63.6(C5'), 70.9 (C3'), 74.6 (C8, 75.7 (OPX C9), 85.2 (C1'), 86.0 (C4'), 91.2(C7), 115.86, 115.91, 116.21, 116.30 (NPx and OPx CH), 122.17 and 122.24(OPx C8a and C9a), 123.50, 123.54, 123.92, 124.05 (NPx and OPx CH),124.65, 124.68 (NPx C8a and C9a), 125.7, 126.3, 126.5, 127.8, 127.9,128.46, 128.58, 128.68, 128.74, 128.98, 129.25, 129.64, 129.76 (NPx andOPx CH), 142.7 (C6), 148.1 (C2), 149.3 (NPx and OPx C1'), 150.4, 150.61,150.66, 150.75 (NPx and OPx C4a and C10a), 161.6 (C4). FABMS m/z 816(M+Na), 794 (M+H). HR-FABMS exact mass found 794.2871, calculated for(C₅₀ H₃₉ N₃ O₇)+H 794.2868.

EXAMPLE 1(h)

N.sup.α -(FIuoren-9-ylmethoxycarbonyl)-Nε-biotinyllysine,Fmoc-Lys(Biotin)-OH (22)

A solution of Et₃ N (698μL, 5.00 mmol) in DMF (70 mL) was added to amixture of the N-hydroxysuccinimidyl ester of biotin (3.41 g, 10.0 mmol)and N.sup.α -Fmoc-lysine 21 (1.84 g, 5.00 mmol), and the resultingmixture was stirred for 6 h, then filtered. Cold aqueous HCl (pH 2, 500mL) was then added and the precipitate was filtered and washed withaqueous HCI (pH2, 3×200 mL) and H₂ O (3×200 mL). The residue was foundto contain a large amount of water at this stage and was consequentlylyophilised (48 h) to give a fluffy colourless solid (2.41 g, 81%), mp181-2° C. (decomp.). ¹ H NMR (d₆ -DMSO): δ1.20-1.40 (m, 4H, Btn Hy andLys Hδ), 1.50-1.52 (m, 6H Btn Hβ and Btn Hδ and Lys Hγ), 1.52-1.62 (m,2H, Lys Hβ), 2.03 (t, 2H, Btn Hα, J=7.3 Hz), 2.56 (d, 1H, Btn H5b,J=12.5 Hz), 2.79 (dd, 1H, Btn H5a, J=12.4, 5.1Hz), 3.00 (m, 2H, LysH11), 3.06 (m, 1H, Btn H2), 3.9 (m, 1H, Lys Hα), 4.1 (m, 1H, Btn H3),4.18-4.30 (m, 4H, Btn H4 and Fmoc CH₂ and Fmoc H9), 6.36 (s, 1H BtnH1'), 6.42 (s, 1H, Btn H3'), 7.32 (ddd, 2H, Fmoc H2 and H7, J=7.4, 7.4,1.1 Hz), 7.41 (dd, 2H, Fmoc H3 and H6, J=7.2, 7.2 Hz), 7.61 (d, 1H, LysN.sup.α H, J=8.1 Hz), 7.72 (d, 2H, Fmoc H1 and H8, J=7.4 Hz), 7.76 (t,1H, Lys N.sup.α H, J=5.6 Hz), 7.88 (d, 2H, Fmoc H4 and H5, J=7.4 Hz). ¹³C NMR (d₆ -DMSO): d 23.1 (Lys Cγ), 25.3 (Btn Cβ), 28.0 (Btn Cδ), 28.2(Btn Cγ), 28.8 (Lys Cδ), 30.5 (Lys Cβ), 35.2 (Btn Cα), 38.2 (Lys Cε),39.9 IBtn C5), 46.7 (Fmoc C9), 53.8 (Lys Cα), 55.2 (Btn C2), 59.2 (BtnC4), 61.0 (Btn C3), 65.6 (Fmoc CH₂), 120.1 (Fmoc C3 and C6), 125.3 (FmocC2 and Cy), 127.1 (Fmoc C4 and C5), 127.7 (Fmoc C1 and C8), 140.7 (FmocC4a and C4b), 143.79, 143.84 (Fmoc C8a and C9a), 156.2 (Fmoc CO), 162.7(Btn C2'), 171.9 (Btn C10), 174.0 (Lys CO₂ H). HR-FABMS exact mass found595.2566, calculated for (C₃₁ H₃₈ N₄ O₆ S)+H 595.2590. IR 1702 cm⁻¹(Fmoc CO and Lysine α CO), 1638 cm⁻¹ (amide CO).

EXAMPLE 1(i)

N.sup.α -(Fluoren-9-ylmethoxycarbonyl)-N.sup.ε -biotinyllysinepentafluoro-phenyl ester, Fmoc-Lys(Biotin)-OPfp (23)

A solution of pentafluorophenol (1.75 g, 9.29 mmol) and DCC (1.27, 6.32mmol) in DMF (5 mL) was added to the solution of 22 (2.21 g, 3.72 mmol)in DMF (40 mL) and the mixture stirred for 16 h. The colourlessprecipitate was filtered off and discarded. The filtrate was kept andafter removal of the solvent under vacuum followed by trituration anddiethyl ether (4×10 mL) to afford a colourless solid which wasrecrystallised from ethyl acetate/ethanol/acetic acid (80:19:1) to givefine colourless crystals (1.90 g, 67%), mp 162-5° (decomp.). ¹ H NMR (d₆DMSO): δ1.23-1.32 (m, 2H, Tbh Hγ), 1.38-1.53 (m, 6H, Btn Hβ and Btn Hδand Lys Hδ), 154-163 (m, 2H, Lys Hβ), 2.04 (t, 2H, Btn Hα: J=7.3 Hz),2.55 (d, 1H, Btn H5b, J=12.5 Hz), 2.79 (dd, 1H, Btn H5a, J=12.5, 5.1Hz), 2.98-3.04 (m, 2H, Lys Hε), 3.05-3.10 (m, 1H, Btn H2), 4.09 (m 1H,Lys αH), 4.20-4.30 (m, 2H. Btn H3 and Fmoc H9), 4.32-4.42 (m, 3H, Btn H4and Fmoc CH₂), 6.35 (s, 1H, Btn H1'), 6.42 (s, 1H, Btn H3') 7.28-7.34(m, 2H, Fmoc H2 and H7, 7.40 (dd, 2H, Fmoc H3 and H6, J=7.5, 7.5 Hz),7.70 (d, 2H, Fmoc H1 and H8, J=7.3 Hz), 7.77 (5, 1H, Lys NeH, J=5.7 Hz),7.88 (d, 2H, Fmoc H4 and H5, J=7.7 Hz), 8.12 (d, 1H Lys N.sup.α H, J=7.3Hz). ¹³ C NMR (d₆ -DMSO): δ22.7 (Lys Cγ), 25.3 (Btn Cβ), 28.1 (Btn Cδ),28.3 (Btn Cγ). 28.7 (Lys Cδ), 29.9 (Lys Cβ), 35.3 (Btn Cα), 38.1 (LysCε), 39.9 (Btn C5), 46.7 (Fmoc C9), 53.9 (Lys Cα), 55.5 (Btn C2), 59.2(Btn C4), 61.1 (Btn C3), 65.9 (Fmoc CH₂), 120.2 (Fmoc C3 and C6), 125.2(Fmoc C2 and C7), 127.1 (Fmoc C4 and C5), 127.7 (Fmoc C1 and C8), 136.5,138.5, 139.0 (2 x m, C-CF), 140.8 (Fmoc C4a and C4b), 142.0 (m, C-CF),143.68, 143.71 (Fmoc C8a and C9a), 156.2 (Fmoc CO), 162.7 (Btn C2'),169.2 (Lys CO), 171.9 (Btn CIO). HR-FABMS exact mass found 761.2413,calculated for (C₃₇ H₃₇ N₄ O₆ SF₅)+H 761.2432. IR 1787 cm⁻¹ (ester CO),1702 cm⁻¹ (Fmoc CO), 1641 cm⁻¹ (amide CO).

EXAMPLE 1(j)

Preparation of Succinyl-CPG resin (16)

A solution of 3-aminopropyltriethoxysilane (3 g, 13.6 mmol) in ethanol(60 mL) was added to CPG (200-400 mesh, 500 Å pore size, Cat. No. 27720)13 (6 g), and the mixture was shaken gently at regular intervals over aperiod of 6 h. The resin was collected by gravity filtration (withoutany washing), air dried (24 h), and kept at 110° C. (24H) to give theaminated resin 14. The amino loading was determined to be 129 μmol/g byninhydrin assay. N-Fmoc-εAhx-OPfp (2.5 molar equiv) and HOBT (2.5 molarequiv) in DMF were coupled to 14 (double coupling, 1.5 h each reaction)in a sintered-glass column. Two or more aminohexanoic acid residues wereattached using 1.5 h single couplings, giving resin 15. The Fmoc groupwas cleaved by treatment with 20% piperidine/DMF (5 min), the resinwashed with DMF, and then a solution of succinic anhydride (50 molarequiv) and DMP (10 molar equiv.) in a minimum volume of dry pyridine wasadded. After shaking the suspension for 1 h, the resin was rinsed withpyridine, DMF, CH₂ Cl₂, and dried under vacuum. The reaction wasmonitored by ninhydrin assay, and the loading of carboxylic acid groupsas calculated from the disappearance of amino groups was 46 μmol/g.

EXAMPLE 1(k)

Coupling of Modified Nucleosides 3 and 4 to Succinyl-CPG Resin 16:

Resin 16 was treated twice with a solution of 3 or 4 (5 molar equiv),diisopropylcarbodiimide (5 molar equiv) and DMAP (0.5 molar equiv) in aminimum volume of dry pyridine in two separate 16 h couplings with onlya washing set up in between (double coupling). After the secondcoupling, the resin was rinsed with pyridine, and pixyl assay gave anucleoside loading of 39 μmol/g. The remaining carboxylic acid and aminogroups were capped with piperidine and Ac₂ O/DMAP according to themethod of Damha⁴, giving resins 18 or 19.

EXAMPLE 1(1)

Derivatisation of 18 and 19 to give 20

In the case of 18, the resin was treated with 90% TFA/ethanedithiol for10 min, rinsed with CH₂ Cl₂, and then neutralised with 20%/a Et₃ N/CH₂Cl₂. The resin was then rinsed with CH₂ Cl₂ dried, and treated with a1:1 mixture of Fmoc-εAhx-OPfp/HOBT in DMF (2.5 molar equiv, 45 min,twice). After rinsing with DMF, the resin was subjected to twosuccessive 16 h reactions with 4,4'-dimethoxytrityl chloride (k50 molarequiv each time) in a minimum volume of dry pyridine to give resin 20.Trityl assay showed 30 μmol/g of dimethoxytrityl group present. Theprocedure for the derivatisation of 19 to give 20 is identical to theprocedure for 18 except that 3% DCA/CH₂ Cl₂ was substituted for 90%TFA/ethanedithiol.

EXAMPLE 1(m)

Polyamide and Oligonucleotide Synthesis on Resin 20

A biotinylated lysine residue and an alanine residue were attached to 20by Fmoc solid phase peptide synthesis, using 23 and N-Fmoc-Ala-OPfprespectively, in a manual glass-sinter peptide synthesis cell. A 5-foldexcess of amino-acid pentafluorophenyl ester and HOBT was used, with acoupling time of one hour and 20% piperidine/DMF as deprotection agent.The α-amino group of alanine was deprotected and capped with Ac₂ O (25μL) and DMAP (0.050 g) in dry pyridine (0.5 h). After rinsing with DMF,CH₂ Cl₂ and drying under vacuum, a portion of the resin was used inoligonucleotide synthesis on an Applied Biosystems 380 A DNASynthesizer' using standard β-cyanoethyl-protected phosphoramidites(with a 60 sec Ac2O/DMAP capping step²), on a 1 μmol scale. The sequenceof the oligonucleotide synthesised was GATGAGTTCGTGTCCGTACAACT* (T*being the modified nucleoside linker). The resulting conjugate wascleaved from the solid support by treatment with concentrated ammonia(22° C., 6h). The resulting solution of the conjugate was heated at 50°C. for 24 h to effect base deprotection. The ammonia was removed underreduced pressure and the conjugate redissolved in 0.1 mM EDTA (2 mL).Separation by preparative PAGE (16% polyacrylamide gel)⁵ showed twoproducts, the one of the lowest electrophoretic mobility was purified togive 5 in an overall yield of 2.1%.

EXAMPLE 1(n)

Characterisation of the Oligonucleotide-Polyamide Conjugate:

A sample of the conjugate 5 was 5'-end labelled⁵ with γ-[³² P]-ATP andT₄ polynucleotide kinase and was shown to be homogeneous by PAGE (16%polyacrylamide gel). The UV spectrum showed a maximum of absorption at260 nm. A 3.0 nmol aliquot of the conjugate (amount calculated from UVabsorption at 260nm) was analysed for amino-acid content, and showed aratio of 1.01 mol Ala to 0.99 mol Lys as expected, the amount of peptidefound in the sample was 2.55 nmol. A 1 μg aliquot of 5 (in 20 μL of H2O)was incubated with P, nuclease (5 μg, in 5 μL of 0.05M NaOAc, pH 6.0)and 0.5M NaOAc (pH 6.0, 2 μL) at 37° C. for 30 min. The resultingenymatic digest was analysed by reverse-phase C₁₈ HPLC, using a lineargradient of 0-100% B over 60 min. The HPLC profile was manuallyintegrated to give nucleotide ratios: pdA (4.67), pdG (5.21), pdC(4.80), pdT (6.05), dG (1.27). Expected ratios: pdA (5.00), pdG (5.00),pdC (5.00), pdT (6.00), dG (1.00).

EXAMPLE 1(o)

Ninhydrin Assay:

This is a modified version of the original assay⁶ which specified anincubation time of 7 min at 100° C. Accurately weighed aliquots of resinwere treated with 76% w/w phenol/ethanol (4 drops from a Pasteurpipette), 0.0002M potassium cyanide/pyridine (8 drops), and 0.28Mninhydrin/ethanol (4 drops) at I 10° C. for 10 min. After dilution with60% ethanol (3.8 mL), the absorptions were measured at 570 nm(ε=15000M-⁻¹ cm⁻¹).

EXAMPLE 1(p)

Pixyl and Trityl Assays

Accurately weighed aliquots of resins were treated with 10%toluenesulfonic acid/acetonitrile (3 mL) for 10 min at room temperature.Absorptions were measured at 445 nm (ε=4400 M⁻¹ cm⁻¹) for pixyl and 507nm (ε=66500 M⁻¹ cm⁻¹) for trityl.

EXAMPLE 1(q)

Fmoc Assay:⁷

Accurately weighed aliquots of resin were treated with piperidine (200μL) and CH₂ Cl₂ (200 μL) for 30 min at room temperature. After dilutionwith CH₂ Cl₂ (3.6 mL), the absorptions were measured at 301 nm (ε=7800M⁻¹ cm⁻¹).

EXAMPLE 2 RESULTS AND DISCUSSION

Synthesis of the Modified Nucleoside

The modified nucleosides 1, 3 and 4 which act as the linker between theoligonucleotide and the polyamide were synthesised in three stepsstarting from commercially-available 5-iodo-2'-deoxyuridine (IDU) 6(Scheme II). The first step involved the preparation of the fourdifferent N-protected propargylamines 9 to 12 by reaction of3-aminopropyne and N-(fluoren-9-ylmethoxycarbonyloxy)succinimide,3-nitropyridine-2-sulfenyl chloride (NPYSCI), di-t-butyldicarbonate and9-chloro-9-phenylxanthene respectively. In the case of the NPYSderivative 10, the presence of a base was necessary for efficientreaction. Triethylamine was found to readily effect nucleophilicsubstitution of the NPYSCI to form the quatemary ammonium salt [(Et₃N)S(C₅ H₃ NO₂)]+Cl⁻, so a 2.5 molar excess of 3-aminopropyne was used toact as base as well. All four protected propargylamines were synthesisedin high yield.

The synthesis of the desired alkynyl nucleosides was attempted by tworoutes: attachment of a protected propargylamine to C5 of the IDUfollowed by protection of the 5'-hydroxyl, and vice-versa. ThePd(O)-catalysed oxidative coupling of a protected aminoalkyne to theunprotected nucleoside resulted in complex mixtures inseparable bysilica gel chromatography. Protection of the 5'-hydroxyl of IDU viaDMP-catalysed alkylation with 9-chloro-9-phenylxanthene gave the5'-protected nucleoside 7 in high yield and purity, and this was coupledwith protected propargylamines 9 to 12, according to the method ofHobbs⁸. The reaction of the protected nucleoside 7 with theFmoc-protected propargylamine 9 produced the desired product 1 in quitelow yields (30%), probably due to partial deprotection of 9 bytriethylamine. The free primary amino groups generated by thedeprotection may also coordinate with the palladium catalyst and hinderthe coupling reaction⁸. The product 1 coeluted with the startingmaterial in silica gel chromatography under a wide range of elutingconditions, and both the ¹ H and ¹³ C NMR spectra showed that itcontained a small amount of 7. The coupling reaction between 7 and theNPYS-protected propargylamine 10 produced a complex mixture which couldonly be resolved by silica gel chromatography followed by C₁₈reverse-phase HPLC. Analysis of the HPLC fractions by ¹ H and ¹³ C NMRspectroscopy and FABMS showed that none contained the desired product.In contrast, the coupling of the Boc and pixyl-protected propargylamines11 and 12 to 7 were straightforward. The Boc-protected nucleoside 3 alsocoelutes with the starting nucleoside 7 but the product 3 could beisolated in high yield nevertheless due to the absence of significantcontaminating amounts of 7. The preparation of the dipicylatednucleoside 4 could be monitored by TLC (5% MeOH/1% Et₃ N/CH₂ Cl₂ and thereaction was judged to be complete after 5 hours. This reaction also hada high yield.

Although the alkynyl nucleosides 3 and 4 have quite complex structures,adequate characterisation could be achieved by 1-dimensional ¹ H and ¹³C NMR spectroscopy, since the multiplets in the former compound are wellseparated and most of the latter's resonances correspond quite closelyto those of analogous compounds we have previously reported³. Somesalient features of the ¹³ C NMR spectrum of these alkynyl nucleosidesare the two resonances in the regions δ73.8-74.6 and δ90.1-91.2corresponding to the alkynyl quaternary carbons C8 and C7 respectively(see Scheme I for numbering system). The downfield shift of C5 fromδ69.9 in IDU to δ98.4-98.7 is consistent with the substitution of aquaternary alkynyl carbon for the iodine atom. All other resonances wereconsistent with the proposed structures of 3 and 4.

The next stage in the synthesis of the conjugate was the derivatisationof a solid support for polyamide and oligonucleotide synthesis.Controlled-pore glass (CPG) resin allows both the polyamide and theoligonucleotide to be synthesised efficiently. Conventional peptidesynthesis resins such as Pepsyn K9 have a higher loading than CPG butefficient DNA synthesis by the phosphoramidite approach is not possible,so CPG is the solid support of choice.

Derivatisation of CPG resin for DNA synthesis is conventionally effectedby amination of the CPG, followed by coupling with a nucleosidesuccinate active ester¹⁰ or a carbodiimide coupling, typically withDCC¹⁰,11. Damha et al., (Nucleic Acids Research (1990), 18, pp.3813-3821) has recently described a method for the attachment ofnucleosides to succinylated CPG resin via a1-(3'-dimethylaninopropyl)-3-ethylcarbodiimide (DEC)-mediatedcondensation. This procedure is more convenient since it obviatesadditional solution-phase manipulation of the nucleoside derivatives.The effectiveness of DEC compared to that of DCC has been attributed toits smaller steric requirement¹¹. With this in mind, we anticipated thatdiisopropylcarbodiimide (DIC) might also be better than DCC. Hence, acomparison of DIC with DCC and DEC was undertaken using a standardnucleoside, 5'-O-dimethoxytrityl-N'-benzoyl-2'-deoxycytidine(dimethoxytrityl=di(p-methoxyphenyl)phenylmethyl). Succinylated-CPGresin was treated with the nucleoside, DMAP (0.5 molar equiv), and theappropriate condensation agent in dry pyridine. After 24 h, the degreeof nucleoside coupling was assessed by trityl assay. The nucleosideloadings were determined to be 22, 18 and 30 μmol/g for DCC, DEC and DICrespectively, C indicating that DIC is the condensation reagent ofchoice.

The solid support for the synthesis of the conjugate was prepared in 4steps starting from CPG resin 13 (Scheme III). The CPG was aminated with3-aminopropyltriethoxysilane¹², and then three 6-aminohexanoic acidspacer residues were attached using standard Fmoc solid-phase peptidesynthesis techniques⁹ to give resin 15. It was anticipated thatincorporation of the three spacer units would allow more efficientoligonucleotide synthesis due to the increased accessibility of theterminal resin-bound nucleoside to reagents, in an analogous manner tolong-chain alkylamine (LCAA) CPG13. The aminated resin 15 was subjectedto DMAP-catalysed succinylation with succinic anhydride to give resin16. Attachment of the appropriate nucleoside to the succinyl resin 16was achieved by DIC/DMAP-mediated condensation, and any free carboxylicacid and amine groups remaining were blocked⁴ byDCC/4-nitrophenol/piperidine and acetic anhydride/DMAP treatmentsrespectively.

The attachment of the Fmoc-nucleoside derivative 1 to the succinyl resin16 was problematic. The use of DMAP is necessary to achieve adequatenucleoside loadings of approximately 30 μmol/g, but we have found¹⁴ thatthe concentration used effects 45% cleavage of the Fmoc group in 24hours. After two successive 24 h coupling reactions between theFmoc-nucleoside I and the succinyl resin 16, comparison of the Fmoc andpixyl assays suggested that about half of the nucleoside had undergoneamine deprotection, and was probably attached to the resin by an amidebond. This side product was not expected to interfere in the preparationof the desired conjugate since it would be expected to be stable to thefinal cleavage conditions and thus stay on the solid support. However,resin derivatized with the Fmoc-nucleoside 1 consistently gave very lowyields of conjugate and was not further investigated.

The Boc and pixyl nucleosides 3 and 4 were readily coupled to the solidsupport 16 under the above conditions, resulting in loadings of 22μmol/g and 40 μmol/g respectively as assessed by pixyl assay. Both theBoc and the pixyl derivatives 3 and 4 gave sufficient nucleosideloadings for efficient oligonucleotide synthesis, but the latter ispreferred due to its facile preparation and the milder deprotectionconditions required.

EXAMPLE 3

Preparation of the Biotinylated Lysine Synthon 23

Previously, we have incorporated biotin residues into conjugates viaglobal biotinylation of the polyamide moiety after deprotection of thee-amino group of the lysine residues¹⁵. This batchwise approach gavelimited control of the placement of the biotins and, in largerpolyamides containing up to 10 lysine residues, where the biotinylationreaction does not go to completion, an uneven distribution of biotinsmay result. The synthon 23 (Scheme IV) allows the incorporation ofbiotins in a highly controlled manner using conventional Fmoc peptidesynthesis. It was prepared in a two-step procedure by biotinylation ofN.sup.α -Fmoc-L-Lys-OH 21 with the N-hydroxysuccinimidyl ester ofbiotin, followed by DCC-mediated condensation with pentafluorophenol. Asynthesis of N-Fmoc-D-Lys(Biofin)-OH has been reported by Jacobson etal.¹⁶, but this method is not practical for large scale synthesesbecause the product is only sparingly soluble in the solvents used forthe extraction step and has a yield of 47% starting from N.sup.α-Fmoc-D-Lys-OH compared to our overall yield of 54% for the active ester23. In addition, only an elemental analysis (with 2.5 equiv of H₂ O and0.5 equiv of DMF as solvents of crystallisation) was given ascharacterisation¹⁶.

Characterisation of the pentafluorophenyl ester 23 was made difficult byits highly complex 1H NMR spectrum and the similarity of its ¹³ C NMRspectrum to that of the starting free acid 22. The only significantdifference in the ¹³ C NMR spectrum of the active ester was the upfieldshift of 5 ppm of the lysine α-carbonyl and the presence of someunresolved multiplets in the aromatic region due to thepentafluorophenyl group. To provide more conclusive evidence for thestructures of both the free acid 22 and the active ester 23, a doublequantum filtered homonuclear shift correlation experiment in thephase-sensitive mode of data accumulation (DQFPh COSY) was performed on22, and a heteronuclear multiple bond connectivity (HMBC) experiment wasundertaken on 23. The cross-peaks in the COSY spectrum of biotinylatedlysine 22 were sufficiently well-resolved to provide unequivocalassignments of all multiplets in the 1-dimensional ¹ H NMR spectrum,even for resonances in the crowded methylene region. The HMBC spectrumof the active ester 23 showed strong connectivities between all thecarbonyls and their adjacent protons except for the resonance at δ169.2,which was tentatively assigned to the α-carbonyl. This had nocorrelation to any protons under these particular experimentalconditions. However, by providing unambiguous assignments for three outof the four resonances in the carbonyl region, the HMBC experimentindirectly confirmed that the resonance at δ169.2 was due to the lysineα-carbonyl, and also confirmed the assignment of the remainingresonances which were basically the addition of the spectra ofN-Fmoc-L-Lys-OH and biotin17. In addition, the IR spectrum of 23 has aband of 1787cm⁻¹ corresponding to the ester carbonyl, a significantshift from the carboxylic acid band of 22 at 1702cm⁻¹.

EXAMPLE 4

Synthesis of the Oligonucleotide-Polyamide Conjugate

The model compound 5 was initially synthesised to test the efficacy ofthese types of conjugates as PCR primers. The polyamide moiety of thisconjugate contains a 6-aminohexanoic acid residue as a spacer (Scheme Iin FIG. 2), in ε-biotinylated lysine residue for detection of the PCRamplification products, and an alanine residue as a reference aminoacid. The oligonucleotide moiety was a 23mer which amplifies a 700base-pair region of the DNA of λ phage; the template is provided withthe standard Cetus PCR kit for control reactions.

As with the synthesis of conjugates we have previously described²,15,the polyamide part of the present conjugate was synthesised first on thederivatised solid support described above, by the conventional Fmocstrategy⁹ since peptide synthesis conditions are harsher above, by theconventional Fmoc strategy9 since peptide synthesis conditions areharsher than those of DNA synthesis. The pendant amino and 5'-hydroxylgroups of the derivatized solid supports 18 and 19 were deprotected bytreatment with 90% TFA/ethanedithiol and 3% DCA/CH₂ Cl₂ respectively andthen neutralised with 20% triethylamine/CH₂ Cl₂. They were thensubjected to a double (45 minute each) coupling with a 1:1 mixture ofN-Fmoc-6-aminohexanoic acid pentafluorophenyl ester (Fmoc-εAhz-OPfp) and1-hydroxybenzotriazole (HOBT) (2.5 molar equiv. of each). Thequalitative trinitrobenzenesulfonic acid test¹⁸ showed only traceamounts of free amino acids at the end of the first coupling and no freeamino groups after the repeat coupling. Reprotection of the 5'-hydroxylwas achieved by tritylation with 4,4'-dimethoxytritylchloride inpyridine (without DMP as catalyst because of the presence of the Fmocgroup) to give resin 20. The trityl loading of the resin after twosuccessive 24 hour treatment was comparable to the nucleoside loadingdetermined prior to the acylation reaction, confirming that theacylation had occurred with a high degree of selectivity at the pendantamine. Any remaining free carboxylic acid and amino groups were blockedas already described.

The biotinylated lysine synthon 23 and Fmoc-Ala-OPfp were coupled tosolid support 20 (Scheme III) using standard Fmoc chemistry with a5-fold excess of amino acid active ester and HOBT. The couplingefficiency of 23 was similar to that of the standard alanine derivative,the reaction being complete in one hour. Amino-acid analysis of thesolid support at this stage gave the expected ratio of lysine toalanine, with loadings of 26 and 23 μmol/g for lysine and alaninerespectively. The Fmoc group of alanine was removed with 20%piperidine/DMF and the resulting free amino group acetylated bytreatment with Ac₂ O/DMAP.

Following the synthesis of the polyamide, the 5'-hydroxyl of theresin-bound nucleoside was deprotected by detritylation with 3% DCA/CH₂Cl₂, and an oligonucleotide was synthesised using standard β-cyanoethylphosphoramidite chemistry²,10,19. The repetitive coupling yields ofoigonucleotide as assessed by trityl assays was comparable with that ofDNA synthesis using normal solid supports. Cleavage from the solidsupport, phosphate and base deprotection by ammonia treatment²,10,12gave a material which was shown to be composed of two major products bypolyacrylamide gel electrophoresis (PAGE). The product with the higherelectrophoretic mobility contained no peptidic material while theslower-moving one contained the desired composition of amino acids.Purification by preparative PAGE resulted in high yields of the desiredconjugate.

The oligonucleotide-polyamide conjugate 5 was characterised by UVspectroscopy, amino-acid analysis, nuclease digestion to its componentnucleotides and PAGE. Quantitation of the oligonucleotide moiety by itsUV absorbance and quantitation of the polyamide moiety by amino acidanalysis gave a 1.2:1 ration of oligonucleotide to polyamide. Inaddition, the reactions of the amino acids were as expected, suggestingthat the polyamide moiety was intact and was stable to oligonucleotidesynthesis conditions. The conjugate was also 5'-end labelled with aradioactive phosphate by γ-[³² P]-ATP and T₄ polynucleotide kinase andanalysed by PAGE with the resulting autoradiogram (FIG. 1) confirmingthe conjugate's homoeneity. Preliminary experiments using this conjugateas a PCR primer gave a product of the expected lengthy. Subsequentchemiluminescent detection of the biotin label in this produce showedunequivocally that the conjugate was incorporated into the PCR productas expected (data will be reported elsewhere). These types of conjugatesare thus viable PCR primers.

EXAMPLE 5

PCR Analysis

The conjugate of Example 4 was dissolved in 0.1 mM EDTA solution to afinal concentration of 100 ng/μL. The sequence of the oligonucleotidewas GATGAGTTCGTGTCCGTACAACT* (where T*=modified deoxyridine bearing thebiotinylated triamide) which when combined with the oligonucleotideprimer GGTTATCGAAATCAGCCACAGCG was used to amplify the 500 bp region7131-7630 of c1857 DNA from the bacteriophage λ, provided with thePerkin-Elmer PCR test kit. The normal oligonucleotide primer (with the3'-nucleotide being a T) was also synthesized and purified in the mannerdescribed above.

Each PCR reaction mixture contained the following: 5 μL of Taqpolymerase solution (2.5 U, in Taq polymerase buffer/50% glycerol), 1 μLof each oligonucleotide primer (100 ng was made up to 50 μL withautoclaved, distilled water and a drop of autoclaved mineral oil wasadded. All reactions were performed on a Perkin Elmer Cetus DNA ThermalCycler instrument, using the following cycle: 95° C. (1 min), 55° C. (1min), 72° C. (1 min), 30 cycles, ending with a 10 min step at 72° C. toensure complete chain elongation. Gel electrophoresis of the PCRreaction mixtures (5 μL) was carried out on an agarose gel (1.5%agarose, 0.001% Ethidium Bromide), with 1 x TBE buffer, at 80 V for 1 h.The agarose gel was blotted onto a nylon membrane by the Southernprocedure. The membrane was then probed for the presence of biotin byusing the PHOTOGENE kit from BRL. Like Technologies Inc.. This detectionis carried out in the following manner.

After soaking the membrane in Tris-buffered saline (TBS Tween 20) andthen blocking in 3% bovine serum albumin in the same buffer, themembrane was incubated for 10 min with a solution of thestreptavidin-alkaline phosphatase conjugate (1 mg/mL in 3M NaCl. 1 mMMgCl₂.0.1 mM ZnCl₂. 30 mM triethanolamine (pH 7.6)) diluted 1:1000 withTBS Tween 20. After washing the membrane with TBS Tween 20 for 15 minand `Final Washer Buffer` (diluted 1:10 with distilled water) for 60 minat room temperature, the membrane was blotted to remove excess buffer.It was then placed in a development folder and treated with thechemiluminescent substrate for alkaline phosphatase supplied by themanufacturer. The membrane was stored in the dark for 3 h after whichtime an X-ray film was placed on top. A strong signal was recorded aftera 30 sec exposure.

The UV visualization of the ethidium bromide-stained gel showed quiteclearly that the PCR using the conjugate as one of the primers gavecomparable amounts of amplified DNA to that performed using only normaloligonucleotide primers. Both PCR products were of the expected length.

To prove unequivocally that the amplification products are due to thebiotinylated conjugate, the products on the agarose gel were blottedinto nylon membrane as described above. This detection system basicallyinvolves the reaction of immobilized biotinylated DNA with astreptavidin-alkaline phosphatase conjugate which in turn catalyses adephosphorylation reaction which results in chemiluminescence. Thechemiluminescence is detected by exposure of the blot to normal X-rayfilm. As expected, there was only one band which contained biotin,corresponding to the PCR products arising from the biotinylatedconjugate PCR primer. This shows unequivocally that the biotinylatedprimer was effectively incorporated into the final amplificationproducts.

Chemiluminescent detection, even of only one biotin in this case, inextremely sensitive. A strong signal resulted after only 30 sec ofexposure time, and there was signal saturation after 5 min.

REFERENCES:

The following references are incorporated herein in their entirety:

1. Becker, J. M.; Wilchek, M., Katchaliski, E., Proc. Nall. A cad. SciUSA 1971, 68. 2604-7.

2. Haralambidis, J.; Duncan, L.; Angus, K.; Tregear, G. W. Nucleic AcidsResearch 1990, 18, 493-499.

3. Haralambidis, J.; Chai, M.; Tregear, G.; Nucleic Acids Research 1987,15, 4857-4876.

4. Damha, M. J.; Giannaris, P. A.; Zabarylo, S. V., Nucleic AcidsResearch 1990, 18, 3813-3821.

5. Penschow, J. P.; Haralambidis, J.; Aldred, P.; Tregear, G. W.;Coghlan, J. P., Methods in Enzymology, 1986, 124, 534-548.

6. Sarin, V. K.; Kent, B. H.; Tarn, J. P.; Merrifield, R. B., AnalyticalBiochemistry, 1981, 117, 147-157; Applied Biosystems Inc. Model 430APeptide Synthesizer User's Manual, Version 1.3B, pp. 7-85 to 7-86, July,1988.

7. Milligen 9050 PepSynthesizer Technical Note 3.10, MilliporeCorporation, 1987.

8. Hobbs Jr., F. W. J Org. Chem., 1989, 54, 3420-3422.

9. Atherton, E.; Sheppard, R. C., Solid Phase Peptide Synthesis--apractical approach, IRL Press: Oxford, 1989.

10. Atkinson, T.; Smith, M. In Oligonucleotide Synthesis: A PracticalApproach; Gait, M. J. Ed.; IRL Press: Oxford, 1984; pp 35-81; Adams, S.P., Kavka, K. S.; Wykes, E. J.; Holder, S. B.; Gallupi, G. R. J Am. ChemSoc. 1983, 105, 661-663.

11. Pon, R. T.; Usman, N.; Ogilvie, K. K. Bio Techniques 1988, 6,768-775.

12. Matteucci, M. D.; Caruthers, M. H. J. Am. Chem. Soc. 1981, 103,3185-3191; Haralarnbidis, J., PhD Thesis, University of Melbourne, 1984.

13. Gait, M. J. Oligonucleotide Synthesis: A Practical Approach; IRLPress: Oxford, 1984.

14. Tong, G. unpublished results.

15. Haralarnbidis, J.; Angus, K.; Pownall, S.; Duncan, L.; Chai, M.;Tregear, G. W. Nucleic Acids Research 1990, 18, 501-505.

16. Jacobson, K. A.; Ukena, D.; Padgett, W.; Kirk, K. L.; Daly, J. W.Biochemical Pharmacology 1987, 36, 1697-1707.

17. Ikura, M.;, Kunio, H., Organic Magnetic Resonance 1982, 20, 266-273.

18. Benjamin, D. M.; McCormack, J. J.; Gump, D. W., Analytical Chemistry1973, 45, 1531-1534.

19. Beaucage, S. L.; Caruthers, M. H., Tetrohedron Lett. 1981, 22,1859-1862.

We claim:
 1. A nucleotide polymer conjugate of the formula (I)

    Nu--NUC--C═C--X.sup.1 --NH--X.sup.2 --X.sup.3          (I)

where, X¹ is an unsubstituted or substituted C₁ -C₁₀ alkylene group, inwhich one or more carbons may optionally be replaced by --NH--, --O-- or--S--, X² is a bond, or an unsubstituted or substituted C₁ -C₂₀ alkylenegroup, in which one or more carbons may optionally be replaced by--NH--, --O-- or --S--, the optional substituents in X¹ or X² areselected from one or more of oxo, amino, thioxo, hydroxyl, mercapto,carboxyl, halogen, lower alkyl, phenyl, amino-lower alkyl, ester-loweralkyl, amido-lower alkyl, ether-lower alkyl, or thioether-lower alkyl,groups, the sulfur analogues of these substituents, or the side-chainsubstituents from naturally occurring amino acids, and the closelyrelated analogues of these sidechains, X³ is an amino acid, or apolyamide linked via its carboxy terminus, NUC is a nucleoside group ofany one of the formulas: ##STR13## where→indicates the bond to the--C.tbd.C-- group in formula (I), and X⁴ is a sugar group of theformula: ##STR14## where the 5' oxygen is linked to Nu, and X⁵ and X⁶are each independently, H or OR, where R is H, a protecting group, or asolid phase matrix, and Nu is an oligonucleotide.
 2. The conjugateaccording to claim 1, whereX¹ is C₁ -C₃ alkylene, X² is --CO--(C₁ to C₉alkylene)--NH--, X³ is a peptide bound through its carboxy terminus, NUCis a nucleoside of the formula: ##STR15## where X⁴, X⁵ and X⁶ are asdefined in claim 1, and Nu has the formula: ##STR16## where B isindependently selected from adenyl, guanyl, thyminyl or cytosinyl, and nis from 1 to about
 400. 3. The conjugate according to claim 2, whereinX¹ is methylene, and X² is --CO--(CH₂)₅ --NH--.
 4. The conjugateaccording to claim 1, which has the formula; ##STR17## where X², X³, X⁵,X⁶ and Nu are as defined in claim
 1. 5. The conjugate according to claim1, wherein X³ is a peptide comprising from 2 to 100 amino acids.
 6. Theconjugate according to claim 1, wherein X³ is a polyamide chaincontaining one or more reporter groups.
 7. The conjugate according toclaim 6, wherein said reporter groups are attached to said chain via theε-amino group on a lysine group present in said chain.
 8. The conjugateaccording to claim 2, where in the definition of Nu, n is from 2 toabout
 200. 9. The conjugate according to claim 1, wherein X⁵ is H, andX⁶ is OR, where R is H or a solid phase matrix.
 10. The conjugateaccording to claim 1, wherein R is a solid phase matrix selected from acontrolled pore glass, or a polystyrene resin.
 11. A process forpreparing a nucleotide polymer conjugate of the formula (I)

    Nu--NUC--C═C--X.sup.1 --NH--X.sup.2 --X.sup.3          (I)

where, X¹ is an unsubstituted or substituted C₁ -C₃₀ alkylene group, inwhich one or more carbons may optionally be replaced by --NH--, --O-- or--S--, X² is a bond, or an unsubstituted or substituted C₁ -C₃₀ alkylenegroup, in which one or more carbons may optionally be replaced by--NH--, --O-- or --S--, the optional substituents in X¹ or X² areselected from one or more of oxo, amino, thioxo, hydroxyl, mercapto,carboxyl, halogen, lower alkyl, phenyl, amino-lower alkyl, ester-loweralkyl, amido-lower alkyl, ether-lower alkyl or thioether-lower alkyl,groups, the sulfur analogues of these substituents, or the side-chainsubstituents from naturally occurring amino acids, and the closelyrelated analogues of these sidechains, X³ is an amino acid, or apolyamide linked via its carboxy terminus, NUC is a nucleoside of anyone of the formulas: ##STR18## where→indicates the bond to the--C.tbd.C-- group in formula (I), and X⁴ is a sugar group of theformula: ##STR19## where the 5' oxygen is linked to Nu, and X⁵ and X⁶are each independently, H or OR, where R is H, a protecting group, or asolid phase matrix, and Nu is a oligonucleotide, which processcomprises: (1) providing a compound of the formula (m): ##STR20## inwhich, NUC' is a group of any one of the following formulas: ##STR21##and X¹, X⁵, and X⁶ are as previously described, and Pr¹ and Pr² areprotecting groups which may be the same or different; (2) deprotectingthe pendant amino group by removing Pr¹ in compound (m) under conditionswhich may or may not remove Pr² and thereafter reacting the deprotectedcompound with a compound of the formula Pr³ XR^(x) wherein X² is aspreviously described, Pr³ is a protecting group and R^(x) is a leavinggroup, so as to covalently link X² to the pendant amino group, to give:##STR22## and in the case where the 5'-OH group is free this group isoptionally reprotected with Pr² a removable protecting group, the sameor different to Pr² in step (1), or when X² is a bond omitting step (2);(3) deprotecting the pendant amino group by removing Pr³, or Pr¹ when X¹is a bond, in the compound of step (2) and reacting it with an activatedamino acid or polyamide, to introduce all or part of X³, and if onlypart of X³ has been introduced, thereafter sequentially adding one ormore activated amino acids or polyamides one or more times understandard peptide synthesis conditions to add the remainder of X³ to thecompound, to form: ##STR23## (4) deprotecting the 5'-OH group of thesugar moiety of the compound of step (3) if not previously deprotectedand reacting the deprotected OH group with an activated nucleotide oroligonucleotide to form a 5'-3' bond, and thereafter sequentially addingone or more activated nucleotides to form an oligonucleotide chain, toadd Nu to the compound; and (5) optionally removing any remainingprotecting groups, and optionally cleaving said compound from a solidphase matrix where X⁵ or X⁶ is OR and R is a solid phase matrix, to givesaid conjugate of the Formula (I).
 12. A process for preparing anucleotide polymer conjugate of the formula: ##STR24## X² is a bond, oran unsubstituted or substituted C₁ -C₂₀ alkylene group, in which one ormore carbons may optionally be replaced by --NH--, --O-- or --S--, theoptional substituents in X² are selected from one or more of oxo, amino,thioxo, hydroxyl, mercapto, carboxyl, halogen, lower alkyl, phenyl,amino-lower alkyl, ester-lower alkyl, amido-lower alkyl, ether-loweralkyl, or thioether-lower alkyl groups, the sulfur analogues of thosesubstituents, or the side-chain substituents from naturally occurringamino acids, and the closely related analogues of these sidechains, X³is an amino acid, or a polyamide linked via its carboxy terminus,X⁵ andX⁶ are each independently, H or OR, where R is H, a protecting group, ora solid phase matrix, and Nu is an oligonucleotide, which processcomprises: (1) providing a compound of the formula (IIIa): ##STR25## inwhich, X⁵ and X⁶ are as previously described, and Pr¹ and Pr² areprotecting groups which may be the same or different; (2) deprotectingthe pendant amino group by removing Pr¹ in compound (IIIa) underconditions which may or may not remove Pr² and thereafter reacting thedeprotected compound with an amino acid of the formula Pr³ X² R^(x)wherein X² is as previously described, Pr³ is a protecting group andR^(x) is a leaving group, so as to covalently link X² to the pendantamino group, and in the case where the 5'-OH group is free this group isoptionally reprotected with a removable protecting group which may bethe same as or different from the protecting group Pr² in step (1), orwhen X² is a bond omitting step (2); (3) deprotecting the pendent aminogroup by removing Pr³, or Pr¹ when X² is a bond, in the compound of step(2) and reacting it with an activated amino acid or polyamide, tointroduce all or part of X³, and if only part of X³ has been introduced,thereafter sequentially adding one or more activated amino acids orpolyamides one or more times under standard peptide synthesis conditionsto add the remainder of X³ to the compound; (4) deprotecting the 5'-OHgroup of the sugar moiety of the compound of step (3), if not previouslydeprotected, and reacting the deprotected OH group with an activatednucleotide or oligonucleotide to form a 5'-3' bond, and thereaftersequentially adding one or more activated nucleotides to form anoligonucleotide chain, to add Nu to the compound; and (5) optionallyremoving any remaining protecting groups, and optionally cleaving saidcompound from a solid phase matrix where X⁵ and X⁶ is OR and R is saidsolid phase matrix, to give said conjugate of the formula (II).
 13. Amethod for determining the presence and location in animal or planttissue of a specific polynucleotide population which comprises:(a)preparing a section of the tissue to be examined; (b) hybridizing thetissue section with an oligonucleotide polymer conjugate according toclaim 1, wherein the oligonucleotide portion of the conjugate iscomplementary to a portion of a target polynucleotide; (c) removingunhybridized probe material from the tissue section; and (d) detectingor identifying the locations in the tissue section where labelling byhybridization of the conjugate has occurred.
 14. A method for detectinga polynucleotide immobilized to or otherwise associated with a supportmatrix, said method comprising contacting the support matrix with anoligonucleotide polymer conjugate according to claim 1, wherein theoligonucleotide portion of the conjugate is complementary to a portionof the target polynucleotide, and thereafter detecting hybridization ofthe conjugate to the support matrix.
 15. A method for detecting thepresence or absence of a specific viral, bacterial or otherpolynucleotide in a biological sample, comprising contacting the nucleicacids of the sample with an oligonucleotide polymer conjugate accordingto claim I which is complementary to a portion of a targetpolynucleotide, and thereafter detecting whether hybridization of theconjugate has occurred.
 16. A diagnostic kit for detecting a desiredpolynucleotide, which comprises an oligonucleotide polymer conjugateaccording to claim 1, wherein the oligonucleotide portion of theconjugate is complementary to a portion of the desired polynucleotide;and reagents for detecting hybridization of the conjugate.
 17. Adiagnostic kit according to claim 16 for use in determination of thepresence and location in animal or plant tissue of a specificpolynucleotide population, which additionally comprises reagents fortissue section preparation.
 18. A method for extending a DNA sequencewhich comprises reacting a conjugate according to claim 1 withnucleotide triphosphates in the presence of DNA or RNA polymerase.
 19. Amethod of amplifying a target DNA or RNA sequence which compriseshybridizing a conjugate according to claim 1 to a target sequence togive a hybridized duplex, incubating the hybridized duplex with DNApolymerase in the presence of nucleotide triphosphate so as to copy thenucleotide sequence of the target sequence, separating the duplex byheat application, and repeating this sequence a plurality of times so asto amplify the number of copies of the target sequence.